We will use a mouse cell line containing a herpes thymidine kinase (tk) gene inserted in the pericentromeric region of a metacentric marker chromosome to study the endogenous properties of mouse satellite DNA. By tracking the tk inserts in this cell line, we hope to understand the mechanisms mediating the fluidity of satellite DNA, and the means whereby satellite DNA exerts position effects on gene expression. Moreover, we also hope to determine if satellite DNA may play a role in providing for centromere function. Thus the specific aims of this grant proposal are: (1) Characterize the tk inserts in the cell line before and after DNA rearrangements by restriction mapping and in situ chromosome hybridizations. This analysis should reveal the mechanisms mediating these rearrangements, i.e. whether they are mediated via sister chromatid exchanges, and/or interchromosomal recombination between nonhomologous chromosomes. (2) Characterize the expression of the tk genes and the chromatin configuration of the tk inserts and the flanking mouse DNA in this cell line and in its tk- and tk+ subclones. These studies should reveal whether the observed position effects on tk expression is at a transcriptional level and if they are accompanied by specific changes in DNA methylation and in chromatin structure. (3) Clone out the tk inserts with some of the flanking mouse DNA sequences and obtain the sequence of the flanking mouse DNA to determine if there are any unusual characteristics that could explain their propensity to rearrange, their ability to mediate position effects on tk gene expression, and their possible role in providing for centromere function. (4) To determine more precisely if there are modifications in the genomic DNA that can account for the activation/inactivation of tk expression, we will carry out genomic sequencing of DNA from tk- and tk+ derivatives of this cell line. (5) Directly determine if satellite DNA sequences can provide for centromere function by microinjecting purified mouse satellite DNA into Xenopus eggs and examining in the electron microscope for the nucleation of kinetochore structures and for the assmebly of microtubular arrays. The health relevance of this proposal lies in the fact that as tumor cells are often associated with DNA rearrangements accompanied by modulation in the expression of oncogenes (i.e. DNA amplifications, chromosomal translocations, etc.), we hope that our studies may in the long term provide insight into the oncogenic process.